title: Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes.
abstract: The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1-D5 and Flt3D1-D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3D1-D4-FL and Flt3D1-D5-FL complexes diffracted to 4.3 and 7.8 Å resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3D1-D4-FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.
authors.affiliation: Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, 9000 Ghent, Belgium.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Verstraete
authors.forename: Bert
authors.initials: B
authors.lastname: Remmerie
authors.forename: Jonathan
authors.initials: J
authors.lastname: Elegheert
authors.forename: Beatrice
authors.initials: B
authors.lastname: Lintermans
authors.forename: Guy
authors.initials: G
authors.lastname: Haegeman
authors.forename: Peter
authors.initials: P
authors.lastname: Vanhoenacker
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051941:fms-Like Tyrosine Kinase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; C084881:flt3 ligand protein; D017351:N-Acetylglucosaminyltransferases; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3